Surgery-induced gut microbial dysbiosis promotes cognitive impairment via regulation of intestinal function and the metabolite palmitic amide.

BACKGROUND: Perioperative neurocognitive disorders (PND) are the most common postoperative complications with few therapeutic options. Gut microbial dysbiosis is associated with neurological diseases; however, the mechanisms by which the microbiota regulates postoperative gastrointestinal and cognitive function are incompletely understood. METHODS: Behavioral testing, MiSeq 16S rRNA gene sequencing, non-target metabolism, intestinal permeability detection, protein assays, and immunofluorescence staining were employed to discern the impacts of surgery on microbial profiles, intestinal barriers, serum metabolism, and the brain. Interventions in mice included fecal microbiota transplantation, the anti-inflammatory agent dexamethasone, Lactobacillus supplementation, indole propionic acid supplementation, and palmitic amide administration. RESULTS: Surgery-induced cognitive impairment occurs predominantly in aged mice, and surgery-induced alterations in the microbiota composition profile exacerbate intestinal barrier disruption in aged mice. These adverse effects can be mitigated by transferring microbiota from young donors or by bolstering the intestinal barrier function using dexamethasone, Lactobacillus, or indole propionic acid. Moreover, microbiota composition profiles can be restored by transplanting feces from young mice to aged surgical mice, improving neuropathology and cognitive function, and these effects coincide with increased intestinal permeability. Metabolomic screening identified alterations in metabolites in mouse serum after surgery, especially the increase in palmitic amide. Palmitic amide levels in serum and brain can be decreased by transplanting feces from young mice to aged surgical mice. Oral palmitic amide exacerbates cognitive impairment and neuropathological changes in mice. CONCLUSIONS: Gut microbial dysbiosis in mice after surgery is a key mechanism leading to cognition dysfunction, which disrupts the intestinal barrier and metabolic abnormalities, resulting in neuroinflammation and dendritic spine loss. Intestinal barrier damage and high level of palmitic amide in old mice may be the cause of high incidence of PND in the elderly. Preoperative microbiota regulation and intestinal barrier restoration may be of therapeutic benefit in preventing PND. Video Abstract.

EPOR activation attenuates colitis via enhancing LC3B-dependent apoptotic cell clearance.

Systemic immune activation and excessive inflammatory response, induced by intestinal barrier damage, are the major characteristics of inflammatory bowel disease (IBD). Excessive apoptotic cell accumulation leads to the production of a large number of inflammatory factors, further aggravating IBD development. Gene set enrichment analysis data showed that the homodimeric erythropoietin receptor (EPOR) was highly expressed in the whole blood of patients with IBD. EPOR is specifically expressed in intestinal macrophages. However, the role of EPOR in IBD development is unclear. In this study, we found that EPOR activation significantly alleviated colitis in mice. Furthermore, in vitro, EPOR activation in bone marrow-derived macrophage (BMDMs) promoted microtubule-associated protein 1 light chain 3B (LC3B) activation and mediated the clearance of apoptotic cells. Moreover, our data showed that EPOR activation facilitated the expression of phagocytosis- and tissue-repair-related factors. Our findings suggest that EPOR activation in macrophages promotes apoptotic cell clearance, probably via LC3B-associated phagocytosis (LAP), providing a new mechanism for understanding pathological progression and a novel potential therapeutic target for colitis.

Promoting AMPK/SR-A1-mediated clearance of HMGB1 attenuates chemotherapy-induced peripheral neuropathy.

BACKGROUND: Chemotherapy-induced peripheral neuropathy (CIPN) is a serious side effect of chemotherapy with poorly understood mechanisms and few treatments. High-mobility group box 1 (HMGB1)-induced neuroinflammation is the main cause of CIPN. Here, we aimed to illustrate the role of the macrophage scavenger receptor A1 (SR-A1) in HMGB1 clearance and CIPN resolution. METHODS: Oxaliplatin (L-OHP) was used to establish a CIPN model. Recombinant HMGB1 (rHMGB1) (his tag) was used to evaluate the phagocytosis of HMGB1 by macrophages. RESULTS: In the clinic, HMGB1 expression and MMP-9 activity were increased in the plasma of patients with CIPN. Plasma HMGB1 expression was positively correlated with the cumulative dose of L-OHP and the visual analog scale. In vitro, engulfment and degradation of rHMGB1 increased and inflammatory factor expression decreased after AMP-activated protein kinase (AMPK) activation. Neutralizing antibodies, inhibitors, or knockout of SR-A1 abolished the effects of AMPK activation on rHMGB1 engulfment. In vivo, AMPK activation increased SR-A1 expression in the dorsal root ganglion, decreased plasma HMGB1 expression and MMP-9 activity, and attenuated CIPN, which was abolished by AMPK inhibition or SR-A1 knockout in the CIPN mice model. CONCLUSION: Activation of the AMPK/SR-A1 axis alleviated CIPN by increasing macrophage-mediated HMGB1 engulfment and degradation. Therefore, promoting HMGB1 clearance may be a potential treatment strategy for CIPN. Video abstract.

Oridonin ameliorates caspase-9-mediated brain neuronal apoptosis in mouse with ischemic stroke by inhibiting RIPK3-mediated mitophagy.

Neuronal loss is a primary factor in determining the outcome of ischemic stroke. Oridonin (Ori), a natural diterpenoid compound extracted from the Chinese herb Rabdosia rubescens, has been shown to exert anti-inflammatory and neuroregulatory effects in various models of neurological diseases. In this study we investigated whether Ori exerted a protective effect against reperfusion injury-induced neuronal loss and the underlying mechanisms. Mice were subjected to transient middle cerebral artery occlusion (tMCAO), and were injected with Ori (5, 10, 20 mg/kg, i.p.) at the beginning of reperfusion. We showed that Ori treatment rescued neuronal loss in a dose-dependent manner by specifically inhibiting caspase-9-mediated neuronal apoptosis and exerted neuroprotective effects against reperfusion injury. Furthermore, we found that Ori treatment reversed neuronal mitochondrial damage and loss after reperfusion injury. In N2a cells and primary neurons, Ori (1, 3, 6 µM) exerted similar protective effects against oxygen-glucose deprivation and reoxygenation (OGD/R)-induced injury. We then conducted an RNA-sequencing assay of the ipsilateral brain tissue of tMCAO mice, and identified receptor-interacting protein kinase-3 (RIPK3) as the most significantly changed apoptosis-associated gene. In N2a cells after OGD/R and in the ipsilateral brain region, we found that RIPK3 mediated excessive neuronal mitophagy by activating AMPK mitophagy signaling, which was inhibited by Ori or 3-MA. Using in vitro and in vivo RIPK3 knockdown models, we demonstrated that the anti-apoptotic and neuroprotective effects of Ori were RIPK3-dependent. Collectively, our results show that Ori effectively inhibits RIPK3-induced excessive mitophagy and thereby rescues the neuronal loss in the early stage of ischemic stroke.

Erythropoietin signaling in peripheral macrophages is required for systemic ß-amyloid clearance.

Impaired clearance of beta-amyloid (Aß) is a primary cause of sporadic Alzheimer's disease (AD). Aß clearance in the periphery contributes to reducing brain Aß levels and preventing Alzheimer's disease pathogenesis. We show here that erythropoietin (EPO) increases phagocytic activity, levels of Aß-degrading enzymes, and Aß clearance in peripheral macrophages via PPARγ. Erythropoietin is also shown to suppress Aß-induced inflammatory responses. Deletion of EPO receptor in peripheral macrophages leads to increased peripheral and brain Aß levels and exacerbates Alzheimer's-associated brain pathologies and behavioral deficits in AD-model mice. Moreover, erythropoietin signaling is impaired in peripheral macrophages of old AD-model mice. Exogenous erythropoietin normalizes impaired EPO signaling and dysregulated functions of peripheral macrophages in old AD-model mice, promotes systemic Aß clearance, and alleviates disease progression. Erythropoietin treatment may represent a potential therapeutic approach for Alzheimer's disease.

Salidroside ameliorates orthopedic surgery-induced cognitive dysfunction by activating adenosine 5'-monophosphate-activated protein kinase signaling in mice.

Perioperative neurocognitive disorders (PND) are the most common postoperative complications with few therapeutic options. Salidroside, a plant-derived compound, has gained increased attention as a treatment for various neurological diseases and particularly as a modifier of microglia-mediated neuroinflammation. However, the effect of salidroside on orthopedic surgery-induced cognitive dysfunction and the underlying mechanisms are largely unknown. Here, we found that salidroside greatly attenuated cognitive impairment in mice after orthopedic surgery. Neuroinflammation in the mouse hippocampus was also attenuated by salidroside. Meanwhile, salidroside treatment induced a switch in microglial polarization to the anti-inflammatory phenotype. In vitro, salidroside suppressed the expression of proinflammatory cytokines and induced a switch in microglial phenotype to the anti-inflammatory phenotype. Mechanistically, molecular docking studies revealed the potential AMPK activation activity of salidroside. And salidroside did up-regulated the AMPK pathway proteins. Moreover, AMPK antagonist abolished the effects of salidroside in vivo and in vitro. Taken together, our results demonstrated that salidroside effectively suppressed PND by suppressing microglia-mediated neuroinflammation through activating AMPK pathway, and it might be a novel therapeutic approach for PND.

Indolepropionic acid reduces obesity-induced metabolic dysfunction through colonic barrier restoration mediated via tuft cell-derived IL-25.

Previous studies have indicated that indolepropionic acid (IPA), derived from dietary tryptophan via gut microbiota conversion, is negatively correlated with type 2 diabetes mellitus and systemic low-grade inflammation. However, the effects of IPA administration on obesity, as well as the underlying mechanisms, remain unclear. In the present study, we observed that obesity leads to a dramatic reduction in IPA levels in both the serum and colonic mucosa, and IPA supplementation exerted beneficial effects on weight, as well as on glucose and lipid metabolism disorders. In adipose tissue, IPA treatment had no direct effect on adipocyte differentiation, but it significantly ameliorated adipose inflammation, thus preventing adipocyte enlargement. Moreover, IPA administration promoted gut integrity, increased the expression of tight junction proteins, and downregulated colonic inflammation; these effects were demonstrated to have a poor relationship with gut microbiota composition. Mechanistically, IPA significantly promoted the expansion of the tuft cell lineage in the gut and increased the secretion of interleukin-25 both in vivo and ex vivo, which contributes to the integrity of the gut barrier. This may partly depend on the free fatty acid receptor 3 pathway in tuft cells. Overall, our results demonstrate that IPA supplementation prevents the development of high-fat diet-induced obesity and metabolic disorders by restoring tuft cell-interleukin-25-mediated colonic barrier integrity; hence, IPA could be a potential agent for treatment of obesity.

Oridonin Ameliorates Traumatic Brain Injury-Induced Neurological Damage by Improving Mitochondrial Function and Antioxidant Capacity and Suppressing Neuroinflammation through the Nrf2 Pathway.

Traumatic brain injury (TBI) is a global public health concern, and few effective treatments for its delayed damages are available. Oridonin (Ori) recently has been reported to show a promising neuroprotective efficacy, but its potential therapeutic effect on TBI has not been thoroughly elucidated. The TBI mouse models were established and treated with Ori or vehicle 30 min post-operation and every 24 h since then. Impairments in cognitive and motor function and neuropathological changes were evaluated and compared. The therapeutic efficacy and mechanisms of action of Ori were further investigated using animal tissues and cell cultures. Ori restored motor function and cognition after TBI-induced impairment and exerted neuroprotective effects by reducing cerebral edema and cortical lesion volume. Ori increased neuronal survival, ameliorating gliosis and the accumulation of macrophages after injury. It suppressed the increased production of reactive oxygen species, lipid peroxide, and malondialdehyde and reversed the decrease of mitochondrial membrane potential and adenosine triphosphate content, which was also identified in oxidatively stressed neuronal cultures. Further, Ori inhibited the expression of nucleotide-binding domain leucine-rich repeats family protein 3 (NLRP3) inflammasome proteins and NLRP3-dependent cytokine interleukin-1ß that can be induced by oxidative stress after TBI. Regarding underlying mechanisms, Ori significantly enhanced expression of key proteins of the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) pathway. Our results demonstrated that Ori effectively improved functional impairments and neuropathological changes in animals with TBI. By activating the Nrf2 pathway, it improved mitochondrial function and antioxidant capacity and suppressed the neuroinflammation induced by oxidative stress. The results therefore suggest Ori as a potent candidate for managing neurological damage after TBI.

Inhibition of Smad3 in macrophages promotes Aß efflux from the brain and thereby ameliorates Alzheimer's pathology.

Impaired amyloid-ß (Aß) clearance is believed to be a primary cause of Alzheimer's disease (AD), and peripheral abnormalities in Aß clearance have recently been linked to AD pathogenesis and progression. Data from recent genome-wide association studies have linked genetic risk factors associated with altered functions of more immune cells to AD pathology. Here, we first identified correlations of Smad3 signaling activation in peripheral macrophages with AD progression and phagocytosis of Aß. Then, manipulating the Smad3 signaling regulated macrophage phagocytosis of Aß and induced switch of macrophage inflammatory phenotypes in our cell cultures. In our mouse models, flag-tagged or fluorescent-dye conjugated Aß was injected into the lateral ventricles or tail veins, and traced. Interestingly, blocking Smad3 signaling efficiently increased Aß clearance by macrophages, reduced Aß in the periphery and thereby enhanced Aß efflux from the brain. Moreover, in our APP/PS1 transgenic AD model mice, Smad3 inhibition significantly attenuated Aß deposition and neuroinflammation, and ameliorated cognitive deficits, probably by enhancing the peripheral clearance of Aß. In conclusion, enhancing Aß clearance by peripheral macrophages through Smad3 inhibition attenuated AD-related pathology and cognitive deficits, which may provide a new perspective for understanding AD and finding novel therapeutic approaches.

CXCR5 Knockdown Attenuates Hippocampal Neurogenesis Deficits and Cognitive Impairment in a Mouse Model of Sepsis-associated Encephalopathy.

In this study, we investigated the potential role of C-X-C chemokine receptor type (CXCR) 5 in neurocognitive function in a mouse model of sepsis-associated encephalopathy (SAE). Adult male C57BL/6J mice received intracerebroventricular injections of small interfering RNAs (siRNAs) against CXCR5 or scrambled control siRNA. After 3 days, SAE was induced by cecal ligation and puncture (CLP, n = 16 per group). Memory and learning ability were tested using the Morris water maze (MWM) on days 5-9 after CLP. Hippocampal expression of CXCR5, interleukin (IL)-1ß and IL-6 were measured by western blot. Cell proliferation and the numbers of immature and mature neurons in the dentate gyrus were assessed by immunohistochemistry. CLP mice had deficits in memory and learning, as shown by increased latency in the MWM training sessions and decreased time spent in and crossing the target quadrant on day 9. CLP also increased the number of proliferating and immature neurons and decreased the number of mature neurons. This was accompanied by increased expression of CXCR5, IL-1ß and IL-6 in the hippocampus. CXCR5 knockdown attenuated the memory and learning deficits induced by CLP and partially reversed the effects of CLP on numbers of proliferating, immature and mature neurons, and on expression of IL-1ß and IL-6 in the hippocampus. These results suggest that CXCR5 knockdown can attenuate sepsis-induced deficits in hippocampal neurogenesis and cognitive function in mice with SAE.

Zerumbone ameliorates behavioral impairments and neuropathology in transgenic APP/PS1 mice by suppressing MAPK signaling.

BACKGROUND: Alzheimer's disease (AD) is a major clinical problem, but there is a distinct lack of effective therapeutic drugs for this disease. We investigated the potential therapeutic effects of zerumbone, a subtropical ginger sesquiterpene, in transgenic APP/PS1 mice, rodent models of AD which exhibit cerebral amyloidosis and neuroinflammation. METHODS: The N9 microglial cell line and primary microglial cells were cultured to investigate the effects of zerumbone on microglia. APP/PS1 mice were treated with zerumbone, and non-cognitive and cognitive behavioral impairments were assessed and compared between the treatment and control groups. The animals were then sacrificed, and tissues were collected for further analysis. The potential therapeutic mechanism of zerumbone and the signaling pathways involved were also investigated by RT-PCR, western blot, nitric oxide detection, enzyme-linked immunosorbent assay, immunohistochemistry, immunofluorescence, and flow cytometry analysis. RESULTS: Zerumbone suppressed the expression of pro-inflammatory cytokines and induced a switch in microglial phenotype from the classic inflammatory phenotype to the alternative anti-inflammatory phenotype by inhibiting the mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B signaling pathway in vitro. After a treatment period of 20 days, zerumbone significantly ameliorated deficits in both non-cognitive and cognitive behaviors in transgenic APP/PS1 mice. Zerumbone significantly reduced ß-amyloid deposition and attenuated pro-inflammatory microglial activation in the cortex and hippocampus. Interestingly, zerumbone significantly increased the proportion of anti-inflammatory microglia among all activated microglia, potentially contributing to reduced ß-amyloid deposition by enhancing phagocytosis. Meanwhile, zerumbone also reduced the expression of key molecules of the MAPK pathway, such as p38 and extracellular signal-regulated kinase. CONCLUSIONS: Overall, zerumbone effectively ameliorated behavioral impairments, attenuated neuroinflammation, and reduced ß-amyloid deposition in transgenic APP/PS1 mice. Zerumbone exhibited substantial anti-inflammatory activity in microglial cells and induced a phenotypic switch in microglia from the pro-inflammatory phenotype to the anti-inflammatory phenotype by inhibiting the MAPK signaling pathway, which may play an important role in its neuroprotective effects. Our results suggest that zerumbone is a potential therapeutic agent for human neuroinflammatory and neurodegenerative diseases, in particular AD.

Suppression of the RAC1/MLK3/p38 Signaling Pathway by ß-Elemene Alleviates Sepsis-Associated Encephalopathy in Mice.

It is still difficult to treat sepsis-associated encephalopathy (SAE) which is a diffuse brain dysfunction caused by sepsis, with excessive activation of microglia as one of the main mechanisms. Ras-related C3 botulinum toxin substrate 1 (RAC1) is proven to be a key molecule in the inflammatory signaling network. By using microglial cell line BV-2 and a mouse model of cecal ligation puncture (CLP), we herein evaluated the effects of ß-elemene, an extract of Curcuma zedoaria Rosc., on RAC1 signaling in microglia. ß-Elemene decreased the expressions of pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6] and attenuated translocation of nuclear factor-κB (NF-κB) p65 from the cytosol to the nucleus in BV-2 cells after lipopolysaccharide (LPS) treatment. It also inhibited the activation of RAC1, mixed-lineage protein kinase 3 (MLK3) and p38 mitogen-activated protein kinase (MAPK). The phosphorylation of the RAC1 Ser71 site was increased by ß-elemene. Moreover, the learning and memory abilities of CLP mice in the water maze test and fear conditioning test were improved after ß-elemene treatment. It reduced the expression of the microglial marker IBA1, significantly increased RAC1 Ser71 phosphorylation, and suppressed the RAC1/MLK3/p38 signaling activation and inflammatory response in the hippocampus. In conclusion, ß-elemene effectively alleviated SAE in mice and inhibited the RAC1/MLK3/p38 signaling pathway in microglia, and might be an eligible potential candidate for SAE treatment.

Down Regulation of the Expression of ELMO3 by COX2 Inhibitor Suppresses Tumor Growth and Metastasis in Non-Small-Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is one of the most common malignancies. Studies have shown that engulfment and cell motility 3 (ELMO3) is highly expressed in NSCLC and can be used as a novel biomarker, but its underlying mechanism remains to be explored. The aim of this study was to investigate the mechanism by which ELMO3 may be down-regulated by COX-2 inhibitors to inhibit NSCLC. NSCLC tissue and adjacent normal lung tissue from 24 patients were used to detect the mRNA and protein expression of ELMO3, COX-2, and other related proteins by Western blot, RT-PCR, and Immunohistochemical analysis. Lewis Lung carcinoma (LLC) cells were used to investigate the effects and the mechanism of siELMO3 and COX-2 inhibitor. C57BL/6 mice inoculated with LLC cells by subcutaneous (s.c.) injection were used to detect the in vivo effects of cox-2 inhibitor. The expression of ELMO3 and cyclooxygenase-2 (COX-2) in human NSCLC tissues was significantly increased compared with that in the adjacent normal tissues. ELMO3 exhibited a positive correlation with COX-2 expression. Moreover, knockdown of ELMO3 suppressed the epithelial-mesenchymal transition (EMT), adhesion, and metastasis of Lewis lung carcinoma (LLC) cells. Importantly, Parecoxib, a selective inhibitor of COX-2, significantly reduced the expression of ELMO3 and EMT in LLC cells and LLC-bearing mice. Furthermore, it could inhibit the growth, adhesion and metastasis of LLC cells in vitro. Our results demonstrate that down regulation of ELMO3 suppressed growth and metastasis of lung cancer by inhibiting EMT. Parecoxib could reduce ELMO3 expression and suppress growth and metastasis of lung cancer, which might be a useful chemotherapeutic agent for inhibiting metastasis and recurrence of NSCLC.

Procyanidins attenuate neuropathic pain by suppressing matrix metalloproteinase-9/2.

BACKGROUND: Management of neuropathic pain is a real clinical challenge. Despite intense investigation, the mechanisms of neuropathic pain remain substantially unidentified. Matrix metalloproteinase (MMP)-9 and MMP-2 have been reported to contribute to the development and maintenance of neuropathic pain. Therefore, inhibition of MMP-9/2 may provide a novel therapeutic approach for the treatment of neuropathic pain. In this study, we aim to investigate the effect of procyanidins (PC), clinically used health product, on MMP-9/2 in neuropathic pain. METHODS: The nociception was assessed by measuring the incidence of foot withdrawal in response to mechanical indentation in mice. Cell signaling was assayed using gelatin zymography, western blotting, and immunohistochemistry. The BV2 cells were cultured to investigate the effects of PC on microglia. RESULTS: Both in vitro and in vivo administration of PC significantly suppresses the activity of MMP-9/2. Oral administration of PC relieves neuropathic pain behaviors induced by chronic constriction sciatic nerve injury (CCI) in mice. Additionally, PC blocks the maturation of interleukin-1ß, which is a critical substrate of MMPs, and markedly suppresses CCI-induced MAPK phosphorylation and neuronal and microglia activation, including the reduced phosphorylation of protein kinase C γ and NMDAR1. Furthermore, PC decreases the phosphorylation of p38 mitogen-activated protein kinase and inhibits the translocation of nuclear factor-κB (NF-κB) in microglia. CONCLUSIONS: PC is an effective and safe approach to alleviate neuropathic pain via a powerful inhibition on the activation of MMP-9/2.

Selective suppression of the JNK-MMP2/9 signal pathway by tetramethylpyrazine attenuates neuropathic pain in rats.

BACKGROUND: Activated astrocytes release matrix metalloproteinase-2/9 (MMP-2/9) to induce central sensitization and maintain neuropathic pain. However, the mechanisms involved in the activation of MMP-2/9 on astrocytes during pain remain poorly understood. Meanwhile, there is a lack of effective treatment to inhibit the activation of MMP-2/9 on astrocytes. In this study, we aim to investigate the effect of tetramethylpyrazine (TMP), a natural compound with analgesic effects but unknown mechanisms, on MMP-2/9 in neuropathic pain. METHODS: The nociception was assessed by measuring the incidence of foot withdrawal in response to mechanical indentation in rats (n = 6). Cell signaling was assayed using western blotting (n = 6) and immunohistochemistry (n = 5). The astrocyte cell line C8-D1A was cultured to investigate the in vitro effects. RESULTS: TMP significantly attenuated the maintenance of chronic constrictive injury (CCI)-induced neuropathic pain, inhibited the activation of astrocytes, and decreased the expression of MMP-2/9. Furthermore, our results indicated that TMP could selectively suppress JNK activity but had no notable effects on ERK and p38. Our study also revealed that the effect of TMP may be dependent on the inhibition of TAK1. CONCLUSIONS: Inhibition of astrocyte activation in the spinal cord by tetramethylpyrazine may have utility in the treatment of CCI-induced neuroinflammation, and our results further implicate JNK-MMP-2/9 as a novel target for the attenuation of neuropathic pain.

AMPK activation by peri-sciatic nerve administration of ozone attenuates CCI-induced neuropathic pain in rats.

Neuropathic pain is a debilitating clinical condition with few efficacious treatments, warranting development of novel therapeutics. Ozone is widely used as an alternative therapy for many different pain conditions, with exact mechanisms still elusive. In this study, we found that a single peri-sciatic nerve injection of ozone decreased mechanical allodynia and thermal hyperalgesia, and normalized the phosphorylation of protein kinase C γ, N-methyl-D-aspartate receptor, and extracellular signal-regulated kinase in a chronic constriction injury (CCI) model in rat sciatic nerve. Meanwhile, ozone significantly suppressed CCI-induced activation of spinal microglia. More importantly, the anti-nociceptive effect of ozone depended on the activation of 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK), which was proved by the fact that the phosphorylated AMPK level increased during the ozone therapy and AMPK antagonist abolished the effect of ozone in vivo and in vitro. In addition, direct injection of AMPK agonist could replicate the anti-nociceptive effect of ozone in CCI rats. In conclusion, our observations indicate that peri-sciatic nerve injection of ozone activates AMPK to attenuate CCI-induced neuropathic pain.

N-acetyl-cysteine attenuates neuropathic pain by suppressing matrix metalloproteinases.

The treatment of neuropathic pain remains a clinical challenge because of its unclear mechanisms and broad clinical morbidity. Matrix metalloproteinase (MMP)-9 and MMP-2 have previously been described as key components in neuropathic pain because of their facilitation of inflammatory cytokine maturation and induction of neural inflammation. Therefore, the inhibition of MMPs may represent a novel therapeutic approach to the treatment of neuropathic pain. In this study, we report that N-acetyl-cysteine (NAC), which is a broadly used respiratory drug, significantly attenuates neuropathic pain through a unique mechanism of MMP inhibition. Both the in vitro (0.1 mM) and in vivo application of NAC significantly suppressed the activity of MMP-9/2. Orally administered NAC (50, 100, and 200 mg/kg) not only postponed the occurrence but also inhibited the maintenance of chronic constrictive injury (CCI)-induced neuropathic pain in rats. The administration of NAC blocked the maturation of interleukin-1ß, which is a critical substrate of MMPs, and markedly suppressed the neuronal activation induced by CCI, including inhibiting the phosphorylation of protein kinase Cγ, NMDAR1, and mitogen-activated protein kinases. Finally, NAC significantly inhibited CCI-induced microglia activation but elicited no notable effects on astrocytes. These results demonstrate an effective and safe approach that has been used clinically to alleviate neuropathic pain through the powerful inhibition of the activation of MMPs.

Activation of Adenosine Monophosphate-activated Protein Kinase Suppresses Neuroinflammation and Ameliorates Bone Cancer Pain: Involvement of Inhibition on Mitogen-activated Protein Kinase.

BACKGROUND: Activation of adenosine monophosphate-activated kinase (AMPK) has been associated with the inhibition of inflammatory nociception and the attenuation of morphine antinociceptive tolerance. In this study, the authors investigated the impact of AMPK activation through resveratrol treatment on bone cancer pain. METHODS: The nociception was assessed by measuring the incidence of foot withdrawal in response to mechanical indentation in rats (n = 8). Cytokine expression was measured using quantitative polymerase chain reaction (n = 8). Cell signalings were assayed by western blot (n = 4) and immunohistochemistry (n = 5). The microglial cell line BV-2, primary astrocytes, and neuron-like SH-SY5Y cells were cultured to investigate the in vitro effects. RESULTS: Resveratrol and 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide, the AMPK activators, significantly attenuated bone cancer pain in rats with tumor cell implantation (TCI; threshold of mechanical withdrawal, resveratrol vs. vehicle: 10.1 ± 0.56 vs. 4.1 ± 0.37; 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide vs. vehicle: 8.2 ± 0.17 vs. 4.1 ± 0.37, mean ± SEM); these effects were reversed by the AMPK inhibitor compound C (compound C vs. resveratrol: 6.2 ± 1.35 vs. 10.1 ± 0.56, mean ± SEM). Resveratrol has an AMPK-dependent inhibitory effect on TCI-evoked astrocyte and microglial activation. The antinociceptive effects of resveratrol were partially mediated by the reduced phosphorylation of mitogen-activated protein kinases and decreased production of proinflammatory cytokines in an AMPK-dependent manner. Furthermore, resveratrol potently inhibited inflammatory factors-mediated protein kinase B/mammalian target of rapamycin signaling in neurons. Acute pain evoked by proinflammatory cytokines in the spinal cord was significantly attenuated by resveratrol. CONCLUSIONS: AMPK activation in the spinal glia by resveratrol may have utility in the treatment of TCI-induced neuroinflammation, and our results further implicate AMPK as a novel target for the attenuation of bone cancer pain.